Poly(ADP-ribose) polymerase-gene

ABSTRACT

The invention relates to poly(ADP-ribose)polymerase (PARP) homologs which have an amino acid sequence which has
     a) a functional NAD +  binding domain and   b) no zinc finger sequence motif of the general formula
 
CX 2 CX m HX 2 C
    in which    m is an integral value from 28 or 30, and the X radicals are, independently of one another, any amino acid;
 
and the functional equivalents thereof; nucleic acids coding therefor; antibodies with specificity for the novel protein; pharmaceutical and gene therapy compositions which comprise products according to the invention; methods for the analytical determination of the proteins and nucleic acids according to the invention; methods for identifying effectors or binding partners of the proteins according to the invention; novel PARP effectors; and methods for determining the activity of such effectors.

The present invention relates to novel poly(ADP-ribose) polymerase(PARP) genes and to the proteins derived therefrom; antibodies withspecificity for the novel proteins; pharmaceutical and gene therapycompositions which comprise products according to the invention; methodsfor the analytical determination of the proteins and nucleic acidsaccording to the invention; methods for identifying effectors or bindingpartners of the proteins according to the invention; methods fordetermining the activity of such effectors and use thereof for thediagnosis or therapy of pathological states.

BACKGROUND OF THE INVENTION

In 1966, Chambon and co-workers discovered a 116 kD enzyme which wascharacterized in detail in subsequent years and is now called PARP (EC2.4.2.30) (poly(adenosine-5′-diphosphoribose) polymerase), PARS(poly(adenosine-5′-diphosphoribose) synthase) or ADPRT(adenosine-5′-diphosphoribose transferase). In the plant kingdom(Arabidopsis thaliana) a 72 kD (637 amino acids) PARP was found in 1995(Lepiniec L. et al., FEBS Lett 1995; 364(2): 103-8). It was not clearwhether this shorter form of PARP is a plant-specific individuality oran artefact (“splice” variant or the like). The 116 kD PARP enzyme hasto date been unique in animals and in man in its activity, which isdescribed below. It is referred to as PARP1 below to avoid ambiguity.

The primary physiological function of PARP 1 appears to be itsinvolvement in a complex repair mechanism which cells have developed torepair DNA strand breaks. The primary cellular response to a DNA strandbreak appears moreover to consist of PARP1-catalyzed synthesis ofpoly(ADP-ribose) from NAD⁺ (cf. De Murcia, G. et al. (1994) TIBS, 19,172).

PARP 1 has a modular molecular structure. Three main functional elementshave been identified to date: an N-terminal 46 kD DNA binding domain; acentral 22 kD automodification domain to which poly(ADP-ribose) becomesattached, with the PARP 1 enzyme activity decreasing with increasingelongation; and a C-terminal 54 kD NAD⁺ binding domain. A leucine zipperregion has been found within the automodification domain, indicatingpossible protein-protein interactions, only in the PARP from Drosophila.All PARPs known to date are presumably active as homodimers.

The high degree of organization of the molecule is reflected in thestrong conservation of the amino acid sequence. Thus, 62% conservationof the amino acid sequence has been found for PARP 1 from humans, mice,cattle and chickens. There are greater structural differences from thePARP from Drosophila. The individual domains themselves in turn haveclusters of increased conservation. Thus, the DNA binding regioncontains two so-called zinc fingers as subdomains (comprising motifs ofthe type CX₂CX_(28/30)HX₂C), which are involved in the Zn²⁺-dependentrecognition of DNA single strand breaks or single-stranded DNA overhangs(e.g. at the chromosome ends, the telomeres). The C-terminal catalyticdomain comprises a block of about 50 amino acids (residues 859-908),which is about 100% conserved among vertebrates (PARP “signature”). Thisblock binds the natural substrate NAD⁺ and thus governs the synthesis ofpoly(ADP-ribose) (cf. de Murcia, loc. cit.). The GX₃GKG motif inparticular is characteristic of PARPs in this block.

The beneficial function described above contrasts with a pathologicalone in numerous diseases (stroke, myocardial infarct, sepsis etc.). PARPis involved in cell death resulting from ischemia of the brain (Choi, D.W., (1997) Nature Medicine, 3, 10, 1073), of the myocardium (Zingarelli,B., et al (1997), Cardiovascular Research, 36, 205) and of the eye (Lam,T. T. (1997), Res. Comm. in Molecular Pathology and Pharmacology, 95, 3,241). PARP activation induced by inflammatory mediators has also beenobserved in septic shock (Szabo, C., et al. (1997), Journal of ClinicalInvestigation, 100, 3, 723). In these cases, activation of PARP isaccompanied by extensive consumption of NAD⁺. Since four moles of ATPare consumed for the biosynthesis of one mole of NAD⁺, the cellularenergy supply decreases drastically. The consequence is cell death.

PARP1 inhibitors described in the abovementioned specialist literatureare nicotinamide and 3-aminobenzamide.3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolone is disclosedby Takahashi, K., et al (1997), Journal of Cerebral Blood Flow andMetabolism 17, 1137. Further inhibitors are described, for example, inBanasik, M., et al. (1992) J. Biol. Chem., 267, 3, 1569 and Griffin, R.J., et al. (1995), Anti-Cancer Drug Design, 10, 507.

High molecular weight binding partners described for human PARP1 includethe base excision repair (BER) protein XRCC1 (X-ray repaircross-complementing 1) which binds via a zinc finger motif and a BRCT(BRCA1 C-terminus) module (amino acids 372-524) (Masson, M., et al.,(1998) Molecular and Cellular Biology, 18,6, 3563).

It is an object of the present invention, because of the diversephysiological and pathological functions of PARP, to provide novel PARPhomologs. The reason for this is that the provision of homologous PARPswould be particularly important for developing novel targets for drugs,and novel drugs, in order to improve diagnosis and/or therapy ofpathological states in which PARP, PARP homologs or substances derivedtherefrom are involved.

BRIEF SUMMARY OF THE INVENTION

We have found that this object is achieved by providing PARP homologs,preferably derived from human and non-human mammals, having an aminoacid sequence which has

-   -   a) a functional NAD⁺ binding domain, i.e. a PARP “signature”        sequence having the characteristic GX₃GKG motif (SEQ ID NO:29);        and    -   b) especially in the N-terminal sequence region, i.e. in the        region of the first 200, such as, for example, in the region of        the first 100, N-terminal amino acids, no PARP zinc finger        sequence motifs of the general formula        CX₂CX_(m)HX₂C (SEQ ID NO:30)    -    in which    -    m is an integral value from 28 or 30, and the X radicals are,        independently of one another, any amino acid;    -   and the functional equivalents thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in more detail withreference to the appended figures. These show:

In FIG. 1 a sequence alignment of human PARP (human PARP1 (SEQ IDNO:35)) and two PARPs preferred according to the invention (human PARP2(SEQ ID NO:2), human PARP3 (SEQ ID NO:6), murine PARP3 (SEQ ID NO:8)),Sequence agreements between human PARP1 and human PARP2, human PARP3 ormurine PARP3 are depicted within frames. The majority sequence (SEQ IDNO:34) is indicated over the alignment. The zinc finger motifs of humanPARP1 are located in the sequence sections corresponding to amino acidresidues 21 to 56 and 125 to 162;

In FIG. 2 Northern blots with various human tissues to illustrate thetissue distribution of PARP2 and PARP3 molecules according to theinvention. Lane 1: brain; lane 2: heart; lane 3: skeletal muscle; lane4: colon; lane 5: thymus; lane 6: spleen; lane 7: kidney; lane 8: liver;lane 9: intestine; lane 10: placenta; lane 11: lung; lane 12: peripheralblood leukocytes; the respective position of the size standard (kb) isindicated.

In FIG. 3 a Northern blot with further various human tissues toillustrate the tissue distribution of the PARP3 molecule according tothe invention. Lane 1: heart; lane 2: brain; lane 3: placenta; lane 4:lung; lane 5: liver; lane 6: skeletal muscle; lane 7: kidney; lane 8:pancreas; the respective position of the size standard (kb) isindicated.

In FIG. 4 a Western blot with various human tissues to illustrate thetissue distribution of the PARP3 molecule according to the invention atthe protein level. Lane 1: heart; lane 2: lung; lane 3: liver; lane 4:spleen; lane 5: kidney; lane 6: colon; lane 7: muscle; lane 8: brain;the respective position of the size standard (kD) is indicated.

In FIG. 5 a Western blot with various human tissues to illustrate thetissue distribution of the PARP3 molecule according to the invention.Lane 1: frontal cortex; lane 2: posterior cortex; lane 3: cerebellum;lane 4: hippocampus; lane 5: olfactory bulb; lane 6: striatum; lane 7:thalamus; lane 8: midbrain; lane 9: entorhinal cortex; lane 10: pons;lane 11: medulla; lane 12: spinal cord.

In FIG. 6 a diagrammatic representation of the PARP assay (ELISA)

In FIG. 7 a diagrammatic representation of the PARP assay (HTRF)

DETAILED DESCRIPTION OF THE INVENTION

Since the PARP molecules according to the invention represent inparticular functional homologs, they naturally also have apoly(ADP-ribose)-synthesizing activity. The NAD binding domainessentially corresponds to this activity and is localized to the Cterminus.

Thus an essential characteristic of the PARPs according to the inventionis the presence of a functional NAD binding domain (PARP signature)which is located in the C-terminal region of the amino acid sequence(i.e. approximately in the region of the last 400, such as, for example,the last 350 or 300, C-terminal amino acids), in combination with anN-terminal sequence having no zinc finger motifs. Since the zinc fingermotifs in known PARPs presumably contribute to recognition of the DNAbreakages, it is to be assumed that the proteins according to theinvention do not interact with DNA or do so in another way. It has beendemonstrated by appropriate biochemical tests that the PARP2 accordingto the invention can be activated by ‘activated DNA’ (i.e. DNA afterlimited DNaseI digestion). It can be concluded from this further thatthe PARP2 according to the invention has DNA binding properties.However, the mechanism of the DNA binding and enzyme activation differsbetween the PARPs according to the invention and PARP1. Its DNA bindingand enzyme activation is, as mentioned, mediated by a characteristiczinc finger motif. No such motifs are present in the PARPs according tothe invention. Presumably these properties are mediated by positivelycharged amino acids in the N-terminal region of the PARPs according tothe invention. Since the ‘activated DNA’ (i.e. for example DNA afterlimited treatment with DNaseI) has a large number of defects (singlestrand breaks, single strand gaps, single-stranded overhangs, doublestrand breaks etc.), it is possible that although PARP1 and the PARPsaccording to the invention are activated by the same ‘activated DNA’, itis by a different subpopulation of defects (e.g. single strand gapsinstead of single strand breaks).

The functional NAD⁺ binding domain (i.e. catalytic domain) binds thesubstrate for poly-(ADP-ribose) synthesis. Consistent with known PARPs,the sequence motif GX¹X²X³GKG (SEQ ID NO:29), in which G is glycine, Kis lysine, and X¹, X² and X³ are, independently of one another, anyamino acid, is present in particular. However, as shown, surprisingly,by comparison of the amino acid sequences of the NAD⁺ binding domains ofPARP molecules according to the invention with previously disclosedhuman PARP1, the sequences according to the invention differ markedlyfrom the known sequence for the NAD⁺ binding domain.

A group of PARP molecules which is preferred according to the inventionpreferably has the following general sequence motif in the catalyticdomain in common:

-   -   PX_(n)(S/T)GX₃GKGIYFA (SEQ ID NO:11), in particular    -   (S/T)XGLR(I/V)XPX_(n)(S/T)GX₃GKGIYFA (SEQ ID NO:12), preferably    -   LLWHG(S/T)X₇IL(S/T)XGLR(I/V)XPX_(n)(S/T)GX₃GKGIYFAX₃SKSAXY (SEQ        ID NO:13)        in which (S/T) describes the alternative occupation of this        sequence position by S or T, (I/V) describes the alternative        occupation of this sequence position by I or V, and n is an        integral value from 1 to 5, and the X radicals are,        independently of one another, any amino acid. The last motif is        also referred to as the “PARP signature” motif.

The automodification domain is preferably likewise present in the PARPsaccording to the invention. It can be located, for example, in theregion from about 100 to 200 amino acids in front of the N-terminal endof the NAD⁺ binding domain.

PARP homologs according to the invention may additionally comprise,N-terminally of the NAD⁺ binding domain (i.e. about 30 to about 80 aminoacids closer to the N terminus), a leucine zipper-like sequence motif ofthe general formula(L/V)X₆LX₆LX₆L (SEQ ID NO:14)in which(L/V) represents the alternative occupation of this sequence position byL or V, and the X radicals are, independently of one another, any aminoacid. The leucine zipper motifs observed according to the inventiondiffer distinctly in position from those described for PARP fromDrosophila. Leucine zippers may lead to homodimers (two PARP molecules)or heterodimers (one PARP molecule with a binding partner differingtherefrom).

The PARP homologs according to the invention preferably additionallycomprise, N-terminally of the abovementioned leucine zipper-likesequence motifs, i.e. about 10 to 250 amino acid residues closer to theN terminus, at least another one of the following part-sequence motifs:LX₉NX₂YX₂QLLX(D/E)X_(b)WGRVG, (motif 1; SEQ ID NO:15)AX₃FXKX₄KTXNXWX₅FX₃PXK, (motif 2; SEQ ID NO:16)QXL(I/L)X₂₁X₉MX₁₀PLGKLX₃QIX₆L, (motif 3; SEQ ID NO:17)FYTXIPHXFGX₃PP, (motif 4; SEQ ID NO:18)andKX₃LX₂LXDIEXAX₂L (motif 5; SEQ ID NO:19),in which (D/E) describes the alternative occupation of this sequenceposition by D or E, (I/L) describes the alternative occupation of thissequence position by I or L, b is the integral value 10 or 11, and the Xradicals are, independently of one another, any amino acid. It is mostpreferred for these motifs 1 to 5 all to be present in the statedsequence, with motif 1 being closest to the N terminus.

The abovementioned PARP signature motif is followed in the proteinsaccording to the invention by at least another one of the followingmotifs:GX₃LXEVALG (motif 6; SEQ ID NO:20)GX₂SX₄GX₃PX_(a)LXGX₂V (motif 7; SEQ ID NO:21) andE(Y/F)X₂YX₃QX₄YLL (motif 8; SEQ ID NO:22)in which (Y/F) describes the alternative occupation of this sequenceposition by Y or F, a is equal to 7 to 9 and X is in each case any aminoacid. It is most preferred for the three C-terminal motifs all to bepresent and in the stated sequence, with motif 8 being closest to the Cterminus.

A preferred PARP structure according to the invention may be describedschematically as follows:

Motifs 1 to 5/PARP signature/motifs 6 to 8 or

motifs 1 to 5/leucine zipper/PARP signature/motifs 6 to 8

it being possible for further amino acid residues, such as, for example,up to 40, to be arranged between the individual motifs and for furtheramino acid residues, such as, for example, up to 80, to be arranged atthe N terminus and/or at the C terminus.

PARP homologs which are particularly preferred according to theinvention are the proteins human PARP2, human PARP3, mouse PARP3 and thefunctional equivalents thereof. The protein referred to as human PARP2comprises 570 amino acids (cf. SEQ ID NO:2). The protein referred to ashuman PARP3 possibly exists in two forms. Type 1 comprises 533 aminoacids (SEQ ID NO:4) and type 2 comprises 540 amino acids (SEQ ID NO:6).The forms may arise through different initiation of translation. Theprotein referred to as mouse PARP3 exists in two forms which differ fromone another by a deletion of 5 amino acids (15 bp). Type 1 comprises 533amino acids (SEQ ID NO: 8) and type 2 comprises 528 amino acids (SEQ IDNO:10). The PARP-homologs of the present invention differ in theirsequences significantly over said PARP protein of Arabidopsis thaliana(see above). For example, PARP2 and PARP3 do not comprise the plant PARPspecific peptide sequence AAVLDQWIPD (SEQ ID NO:31), corresponding toamino acid residues 143 to 152 of the Arabidopsis protein.

The invention further relates to the binding partners for the PARPhomologs according to the invention. These binding partners arepreferably selected from

-   a) antibodies and fragments such as, for example, Fv, Fab, F(ab′)₂,    thereof-   b) protein-like compounds which interact, for example via the above    leucine zipper region or another sequence section, with PARP, and-   c) low molecular weight effectors which modulate a biological PARP    function such as, for example, the catalytic PARP activity, i.e.    NAD⁺-consuming ADP ribosylation, or the binding to an activator    protein or to DNA.

The invention further relates to nucleic acids comprising

-   a) a nucleotide sequence coding for at least one PARP homolog    according to the invention, or the complementary nucleotide sequence    thereof;-   b) a nucleotide sequence which hybridizes with a sequence as    specified in a), preferably under stringent conditions; or-   c) nucleotide sequences which are derived from the nucleotide    sequences defined in a) and b) through the degeneracy of the genetic    code.

Nucleic acids which are suitable according to the invention comprise inparticular at least one of the partial sequences which code for theabovementioned amino acid sequence motifs.

Nucleic acids which are preferred according to the invention comprisenucleotide sequences as shown in SEQ ID NO: 1 and 3, and, in particular,partial sequences thereof which are characteristic of PARP homologsaccording to the invention, such as, for example, nucleotide sequencescomprising

a) nucleotides +3 to +1715 shown in SEQ ID NO:1;

b) nucleotides +242 to +1843 shown in SEQ ID NO:3;

c) nucleotides +221 to +1843 shown in SEQ ID NO:5;

d) nucleotides +112 to +1710 shown in SEQ ID NO:7; or

e) nucleotides +1 to +1584 shown in SEQ ID NO:9

or partial sequences of a), b), c), d) and e) which code for theabovementioned characteristic amino acid sequence motifs of the PARPhomologs according to the invention.

The invention further relates to expression cassettes which comprise atleast one of the above-described nucleotide sequences according to theinvention under the genetic control of regulatory nucleotide sequences.These can be used to prepare recombinant vectors according to theinvention, such as, for example, viral vectors or plasmids, whichcomprise at least one expression cassette according to the invention.

Recombinant microorganisms according to the invention are transformedwith at least one of the abovementioned vectors.

The invention also relates to transgenic mammals transfected with avector according to the invention.

The invention further relates to an in vitro detection method, which canbe carried out homogeneously or heterogeneously, for PARP inhibitors,which comprises

-   a) incubating an unsupported or supported poly-ADP-ribosylatable    target with a reaction mixture comprising    -   a1) a PARP homolog according to the invention;    -   a2) a PARP activator; and    -   a3) a PARP inhibitor or an analyte in which at least one PARP        inhibitor is suspected;-   b) carrying out the polyADP ribosylation reaction; and-   c) determining the polyADP ribosylation of the target qualitatively    or quantitatively.

The detection method is preferably carried out by preincubating the PARPhomolog with the PARP activator and the PARP inhibitor or an analyte inwhich at least one PARP inhibitor is suspected, for example for about1-30 minutes, before carrying out the poly-ADP ribosylation reaction.

After activation by DNA with single strand breaks (referred to as“activated DNA” according to the invention), PARP poly-ADP ribosylates alarge number of nuclear proteins in the presence of NAD. These proteinsinclude, on the one hand, PARP itself, but also histones etc.

The poly-ADP-ribosylatable target preferably used in the detectionmethod is a histone protein in its native form or apolyADP-ribosylatable equivalent derived therefrom. A histonepreparation supplied by Sigma (SIGMA, catalogue No. H-7755; histone typeII-AS from calf thymus, Luck, J. M., et al., J. Biol. Chem., 233, 1407(1958), Satake K., et al., J. Biol. Chem., 235, 2801 (1960)) was used byway of example. It is possible in principle to use all types of proteinsor parts thereof amenable to polyADP-ribosylation by PARP. These arepreferably nuclear proteins, e.g. histones, DNA polymerase, telomeraseor PARP itself. Synthetic peptides derived from the correspondingproteins can also act as target.

In the ELISA according to the invention it is possible to use amounts ofhistones in the range from about 0.1 μg/well to about 100 μg/well,preferably about 1 μg/well to about 10 μg/well. The amounts of the PARPenzyme are in a range from about 0.2 pmol/well to about 2 nmol/well,preferably from about 2 pmol/well to about 200 pmol/well, the reactionmixture comprising in each case 100 μg/well. Reductions to smaller wellsand correspondingly smaller reaction volumes are possible.

In the HTRF assay according to the invention, identical amounts of PARPare employed, and the amount of histone or modified histones is in therange from about 2 ng/well to about 25 μg/well, preferably about 25ng/well to about 2.5 μg/well, the reaction mixture comprising in eachcase 50 μl/well. Reductions to smaller wells and correspondingly smallerreaction volumes are possible.

The PARP activator used according to the invention is preferablyactivated DNA.

Various types of damaged DNA can function as activator. DNA damage canbe produced by digestion with DNases or other DNA-modifying enzymes(e.g. restriction endonucleases), by irradiation or other physicalmethods or chemical treatment of the DNA. It is further possible tosimulate the DNA damage situation in a targeted manner using syntheticoligonucleotides. In the assays indicated by way of example, activatedDNA from calf thymus was employed (Sigma, product No. D4522; CAS:91080-16-9, prepared by the method of Aposhian and Kornberg using calfthymus DNA (SIGMA D-1501) and deoxyribonuclease type I (D-4263).Aposhian H. V. and Kornberg A., J. Biol. Chem., 237, 519 (1962)). Theactivated DNA was used in a concentration range from 0.1 to 1000 μg/ml,preferably from 1 to 100 μg/ml, in the reaction step.

The polyADP ribosylation reaction is started in the method according tothe invention by adding NAD⁺. The NAD concentrations were in a rangefrom about 0.1 μM to about 10 mM, preferably in a range from about 10 μMto about 1 mM.

In the variant of the above method which can be carried outheterogeneously, the polyADP ribosylation of the supported target isdetermined using anti-poly(ADP-ribose) antibodies. To do this, thereaction mixture is separated from the supported target, washed andincubated with the antibody. This antibody can itself be labeled.However, as an alternative for detecting bound antipoly(ADP-ribose)antibody a labeled secondary antibody or a corresponding labeledantibody fragment may be applied. Suitable labels are, for example,radiolabeling, chromophore- or fluorophore-labeling, biotinylation,chemiluminescence labeling, labeling with paramagnetic material or, inparticular, enzyme labels, e.g. with horseradish peroxidase. Appropriatedetection techniques are generally known to the skilled worker.

In the variant of the above process which can be carried outhomogeneously, the unsupported target is labeled with an acceptorfluorophore. The target preferably used in this case is biotinylatedhistone, the acceptor fluorophore being coupled via avidin orstreptavidin to the biotin groups of the histone. Particularly suitableas acceptor fluorophore are phycobiliproteins (e.g. phycocyanins,phycoerythrins), e.g. R-phycocyanin (R-PC), allophycocyanin (APC),R-phycoerythrin (R-PE), C-phycocyanin (C-PC), B-phycoerythrin (B-PE) ortheir combinations with one another or with fluorescent dyes such asCy5, Cy7 or Texas Red (Tandem system) (Thammapalerd, N. et al.,Southeast Asian Journal of Tropical Medicine & Public Health, 27(2):297-303 (1996); Kronick, M. N. et al., Clinical Chemistry, 29(9),1582-1586 (1986); Hicks, J. M., Human Pathology, 15(2), 112-116 (1984)).The dye XL665 used in the examples is a crosslinked allophycocyanin(Glazer, A. N., Rev. Microbiol., 36, 173-198 (1982); Kronick, M. N., J.1 mm. Meth., 92, 1-13 (1986); MacColl, R. et al., Phycobiliproteins, CRCPress, Inc., Boca Raton, Fla. (1987); MacColl, R. et al., Arch. Biochem.Biophys., 208(1), 42-48 (1981)).

It is additionally preferred in the homogeneous method to determine thepolyADP ribosylation of the unsupported target usinganti-poly(ADP-ribose) antibody which is labeled with a donor fluorophorewhich is able to transfer energy to the acceptor fluorophore when donorand acceptor are close in space owing to binding of the labeled antibodyto the polyADP-ribosylated histone. A europium cryptate is preferablyused as donor fluorophore for the anti-poly(ADP-ribose) antibody.

Besides the europium cryptate used, other compounds are also possible aspotential donor molecules. This may entail, on the one hand,modification of the cryptate cage. Replacement of the europium by otherrare earth metals such as terbium is also conceivable. It is crucialthat the fluorescence has a long duration to guarantee the time delay(Lopez, E. et al., Clin. Chem. 39/2, 196-201 (1993); U.S. Pat. No.5,534,622).

The detection methods described above are based on the principle thatthere is a correlation between the PARP activity and the amount ofADP-ribose polymers formed on the histones. The assay described hereinmakes it possible to quantify the ADP-ribose polymers using specificantibodies in the form of an ELISA and an HTRF (homogenous time-resolvedfluorescence) assay. Specific embodiments of these two assays aredescribed in detail in the following examples.

The developed HTRF (homogeneous time-resolved fluorescence) assay systemmeasures the formation of poly(ADP-ribose) on histones using specificantibodies. In contrast to the ELISA, this assay is carried out inhomogeneous phase without separation and washing steps. This makes ahigher sample throughput and a smaller susceptibility to errorspossible. HTRF is based on the fluorescence resonance energy transfer(FRET) between two fluorophores. In a FRET assay, an excited donorfluorophore can transfer its energy to an acceptor fluorophore when thetwo are close to one another in space. In HTRF technology, the donorfluorophore is a europium cryptate [(Eu)K] and the acceptor is XL665, astabilized allophycocyanin. The europium cryptate is based on studies byJean Marie Lehn (Strasbourg) (Lopez, E. et al., Clin. Chem. 39/2,196-201 (1993); U.S. Pat. No. 5,534,622).

In a homogeneous assay, all the components are also present during themeasurement. Whereas this has advantages for carrying out the assay(rapidity, complexity), it is necessary to preclude interference byassay components (inherent fluorescence, quenching by dyes etc.). HTRFprecludes such interference by time-delayed measurement at twowavelengths (665 nm, 620 nm). The HTRF has a very long decay time andtime-delayed measurement is therefore possible. There is no longer anyinterference from short-lived background fluorescence (e.g. from assaycomponents or inhibitors of the substance library). In addition,measurement is always carried out at two wavelengths in order tocompensate for quench effects of colored substances. HTRF assays can becarried out, for example, in 96- or 384-well microtiter plate format andare evaluated using a discovery HTRF microplate analyzer (Can berraPackard).

Also provided according to the invention are the following in vitroscreening methods for binding partners for PARP, in particular for aPARP homolog according to the invention.

A first variant is carried out by

-   a1) immobilizing at least one PARP homolog on a support;-   b1) contacting the immobilized PARP homolog with an analyte in which    at least one binding partner is suspected; and-   c1) determining, where appropriate after an incubation period,    analyte constituents bound to the immobilized PARP homolog.

A second variant entails

-   a2) immobilizing on a support an analyte which comprises at least    one possible binding partner for the PARP homolog;-   b2) contacting the immobilized analyte with at least one PARP    homolog for which a binding partner is sought; and-   c3) examining the immobilized analyte, where appropriate after an    incubation period, for binding of the PARP homolog.

The invention also relates to a method for the qualitative orquantitative determination of a nucleic acid encoding a PARP homolog,which comprises

-   a) incubating a biological sample with a defined amount of an    exogenous nucleic acid according to the invention (e.g. with a    length of about 20 to 500 bases or longer), hybridizing, preferably    under stringent conditions, determining the hybridizing nucleic    acids and, where appropriate, comparing with a standard; or-   b) incubating a biological sample with a defined amount of    oligonucleotide primer pairs with specificity for a PARP    homolog-encoding nucleic acid, amplifying the nucleic acid,    determining the amplification product and, where appropriate,    comparing with a standard.

The invention further relates to a method for the qualitative orquantitative determination of a PARP homolog according to the invention,which comprises

-   a) incubating a biological sample with at least one binding partner    specific for a PARP homolog,-   b) detecting the binding partner/PARP complex and, where    appropriate,-   c) comparing the result with a standard.

The binding partner in this case is preferably an anti-PARP antibody ora binding fragment thereof, which carries a detectable label whereappropriate.

The determination methods according to the invention for PARP, inparticular for PARP homologs and for the coding nucleic acid sequencesthereof, are suitable and advantageous for diagnosing sepsis- orischemia-related tissue damage, in particular strokes, myocardialinfarcts, diabetes or septic shock.

The invention further comprises a method for determining the efficacy ofPARP effectors, which comprises

-   a) incubating a PARP homolog according to the invention with an    analyte which comprises an effector of a physiological or    pathological PARP activity; removing the effector again where    appropriate; and-   b) determining the activity of the PARP homolog, where appropriate    after adding substrates or cosubstrates.

The invention further relates to gene therapy compositions whichcomprise in a vehicle acceptable for gene therapy a nucleic acidconstruct which

-   a) comprises an antisense nucleic acid against a coding nucleic acid    according to the invention; or-   b) a ribozyme against a noncoding nucleic acid according to the    invention; or-   c) codes for a specific PARP inhibitor.

The invention further relates to pharmaceutical compositions comprising,in a pharmaceutically acceptable vehicle, at least one PARP proteinaccording to the invention, at least one PARP binding partner accordingto the invention or at least one coding nucleotide sequence according tothe invention.

Finally, the invention relates to the use of binding partners of a PARPhomolog for the diagnosis or therapy of pathological states in thedevelopment and/or progress of which at least one PARP protein, inparticular a PARP homolog according to the invention, or a polypeptidederived therefrom, is involved. The binding partner used can be, forexample, a low molecular weight binding partner whose molecular weightcan be, for example, less than about 2000 dalton or less than about 1000dalton.

The invention additionally relates to the use of PARP binding partnersfor the diagnosis or therapy of pathological states mediated by anenergy deficit. An energy deficit for the purpose of the presentinvention is, in particular, a cellular energy deficit which is to beobserved in the unwell patient systemically or in individual bodyregions, organs or organ regions, or tissues or tissue regions. This ischaracterized by an NAD and/or ATP depletion going beyond (above orbelow) the physiological range of variation of the NAD and/or ATP leveland mediated preferably by a protein with PARP activity, in particular aPARP homolog according to the invention, or a polypeptide derivedtherefrom.

“Energy deficit-mediated disorders” for the purpose of the inventionadditionally comprise those in which tissue damage is attributable tocell death resulting from necrosis or apoptosis. The methods accordingto the invention are suitable for treating and preventing tissue damageresulting from cell damage due to apoptosis or necrosis; damage to nervetissue due to ischemias and/or reperfusion; neurological disorders;neurodegenerative disorders; vascular stroke; for treating andpreventing cardiovascular disorders; for treating other disorders orconditions such as, for example, age-related macular degeneration, AIDSor other immunodeficiency disorders; arthritis; atherosclerosis;cachexia; cancer; degenerative disorders of the skeletal muscles;diabetes; cranial trauma; inflammatory disorders of the gastrointestinaltract such as, for example, Crohn's disease; muscular dystrophy;osteoarthritis; osteoporosis; chronic and/or acute pain; kidney failure;retinal ischemia; septic shock (such as, for example, endotoxin shock);aging of the skin or aging in general; general manifestations of aging.The methods according to the invention can additionally be employed forextending the life and the proliferative capacity of body cells and forsensitizing tumor cells in connection with irradiation therapy.

The invention particularly relates to the use of a PARP binding partneras defined above for the diagnosis or therapy (acute or prophylactic) ofpathological states mediated by energy deficits and selected fromneurodegenerative disorders, or tissue damage caused by sepsis orischemia, in particular of neurotoxic disturbances, strokes, myocardialinfarcts, damage during or after infarct lysis (e.g. with TPA, Reteplaseor mechanically with laser or Rotablator) and of microinfarcts duringand after heart valve replacement, aneurysm resections and hearttransplants, trauma to the head and spinal cord, infarcts of the kidney(acute kidney failure, acute renal insufficiency or damage during andafter kidney transplant), damages of skeletal muscle, infarcts of theliver (liver failure, damage during or after a liver transplant),peripheral neuropathies, AIDS dementia, septic shock, diabetes,neurodegenerative disorders occurring after ischemia, trauma(craniocerebral trauma), massive bleeding, subarachnoid hemorrhages andstroke, as well as neurodegenerative disorders like Alzheimer's disease,multi-infarct dementia, Huntington's disease, Parkinson's disease,amyotrophic lateral sclerosis, epilepsy, especially of generalizedepileptic seizures such as petit mal and tonoclonic seizures and partialepileptic seizures, such as temporal lobe, and complex partial seizures,kidney failure, also in the chemotherapy of tumors and prevention ofmetastasis and for the treatment of inflammations and rheumaticdisorders, e.g. of rheumatoid arthritis; further for the treatment ofrevascularization of critically narrowed coronary arteries andcritically narrowed peripheral arteries, e.g. leg arteries.

“Ischemia” comprises for the purposes of the invention a localizedundersupply of oxygen to a tissue, caused by blockage of arterial bloodflow. Global ischemia occurs when the blood flow to the entire brain isinterrupted for a limited period. This may be caused, for example, bycardiac arrest. Focal ischemia occurs when part of the brain is cut offfrom its normal blood supply. Focal ischemia may be caused bythromboembolic closure of a blood vessel, by cerebral trauma, edemas orbrain tumor. Even transient ischemias can lead to wideranging neuronaldamage. Although damage to “nerve tissue” may occur days or weeks afterthe start of the ischemia, some permanent damage (e.g. necrotic celldeath) occurs in the first few minutes after interruption of the bloodsupply. This damage is caused, for example, by the neurotoxicity ofglutamate and follows secondary reperfusion, such as, for example,release of free radicals (e.g. oxygen free radicals, NO free radicals).Ischemias may likewise occur in other organs and tissues such as, forexample, in the heart (myocardial infarct and other cardiovasculardisorders caused by occlusion of the coronary arteries) or in the eye(ischemia of the retina).

The invention additionally relates to the use of an effectivetherapeutic amount of a PARP binding partner for influencing neuronalactivity. “Neuronal activity” for the purposes of the invention mayconsist of stimulation of damaged neurons, promotion of neuronalregeneration or treatment of neuronal disorders.

“Neuronal damage” for the purposes of the invention comprises every typeof damage to “nerve tissue” and every physical or mental impairment ordeath resulting from this damage. The cause of the damage may be, forexample, metabolic, toxic, chemical or thermal in nature and includes byway of example ischemias, hypoxias, trauma, cerebrovascular damage,operations, pressure, hemorrhages, irradiation, vasospasms,neurodegenerative disorders, infections, epilepsy, perception disorders,disturbances of glutamate metabolism and the secondary effects causedthereby.

“Nerve tissue” for the purposes of the invention comprises the variouscomponents forming the nervous system, consisting of, inter alfa,neurons, glia cells, astrocytes, Schwann cells, the vascular systeminside and for supplying, the CNS, brain, brain stem, spinal cord,peripheral nervous system etc.

“Neuroprotective” for the purposes of the invention comprises thereduction, the cessation, the slowing down or the improvement ofneuronal damage and the protection, the restoration and the regenerationof nerve tissue which was exposed to neuronal damage.

“Prevention of neurodegenerative disorders” includes the possibility ofpreventing, slowing down and improving neurodegenerative disorders inpeople for whom such a disorder has been diagnosed or who are includedin appropriate risk groups for these neurodegenerative disorders.Treatments for people already suffering from symptoms of these disordersare likewise meant.

“Treatment” for the purposes of the invention comprises

-   (i) preventing a disorder, a disturbance or a condition in people    with a predisposition thereto;-   (ii) preventing a disorder, a disturbance or a condition by slowing    down its advance; and-   (iii) improving a disorder, a disturbance or a condition.

Examples of “neurological disorders” which can be treated by the methodsaccording to the invention are neuralgias (trigeminal,glossopharyngeal), myasthenia gravis, muscular dystrophies, amyorophiclateral sclerosis (ALS), progressive muscular atrophy, peripheralneuropathies caused by poisoning (e.g. lead poisoning), Guillain-Barrésyndrome, Huntington's disease, Alzheimer's disease, Parkinson'sdisease, or plexus disorders. The methods according to the invention arepreferably suitable for treating neurological disorders selected fromperipheral neuropathies caused by physical injury or illness; cranialtrauma such as, for example, traumatic brain injury; physical damage tothe spinal cord; stroke associated with brain damage, such as vascularstroke in conjunction with hypoxia and brain damage, and cerebralreperfusion damage; demyelinating disorders (myelopathies, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis).

The methods according to the invention can additionally be used fortreating cardiovascular disorders. “Cardiovascular disorders” for thepurposes of the invention comprise those which cause ischemias or arecaused by ischemias or ischemia/reperfusion of the heart. Examples arecoronary vessel disorders (for example. atherosclerosis), anginapectoris, myocardial infarct, cardiovascular damage due to cardiacarrest or bypass operation.

The methods according to the invention can be used for treating canceror for sensitizing cancer cells for irradiation therapy. The term“cancer” is to be understood in the widest sense. Modulators of theproteins according to the invention can be used as “anti-cancer therapyagents”. For example, the methods can be used for treating types ofcancer or tumor cells, such as ACTHproducing tumors, acute lymphatic orlymphoblastic leukemia; acute or chronic lymphocytic leukemia; acutenonlymphocytic leukemia; bladder cancer; brain tumors; breast cancer;cervical carcinoma; chronic myelocytic leukemia; bowel cancer; T-zonelym-phoma; endometriosis; esophageal cancer; gall bladder cancer;Ewing's sarcoma; head and neck cancer; cancer of the tongue; Hodgkin'slymphoma; Kaposi's sarcoma; renal cancer; liver cancer; lung cancer;mesothelioma; multiple myeloma; neuroblastoma; nonHodgkin lymphoma;osteosarcoma; ovarian carcinoma; glioblastoma; mammary carcinoma;cervical carcinoma; prostate cancer; pancreatic cancer; penis cancer;retinoblastoma; skin cancer; stomach cancer; thyroid cancer; uterinecarcinoma; vaginal carcinoma; Wilm's tumor; or trophoblastoma.

“Radiosensitizer” or “irradiation sensitizer” for the purposes of theinvention relates to molecules which increase the sensitivity of thecells in the body to irradiation with electromagnetic radiation (forexample X-rays) or speed up this irradiation treatment. Irradiationsensitizers increase the sensitivity of cancer cells to the toxiceffects of the electromagnetic radiation. Those disclosed in theliterature include mitomycin C, 5-bromodeoxyuridine and metronidazole.It is possible to use radiation with wavelengths in the range from 10⁻²⁰to 10 meters, preferably gamma rays (10⁻²⁰ to 10⁻¹³ m), X-rays (10⁻¹¹ to10⁻⁹ m), ultraviolet radiation (10 nm to 400 nm), visible light (400 nmto 700 nm), infrared radiation (700 nm to 1 mm) and microwave radiation(1 mm to 30 cm).

Disorders which can be treated by such a therapy are, in particular,neoplastic disorders, benign or malignant tumors and cancer.

The treatment of other disorders using electromagnetic radiation islikewise possible.

The present invention will now be described in more detail withreference to the appended figures. These show:

Further preferred embodiments of the invention are described in thefollowing sections.

PARP Homologs and Functional Equivalents

Unless stated otherwise, for the purposes of the present descriptionamino acid sequences are indicated starting with the N terminus. If theone-letter code is used for amino acids, then G is glycine, A isalanine, V is valine, L is leucine, I is isoleucine, S is serine, T isthreonine, D is aspartic acid, N is asparagine, E is glutamic acid, Q isglutamine, W is tryptophan, H is histidine, R is arginine, P is proline,K is lysine, Y is tyrosine, F is phenylalanine, C is cysteine and M ismethionine.

The present invention is not confined to the PARP homologs specificallydescribed above. On the contrary, those homologs which are functionalequivalents thereof are also embraced. Functional equivalents compriseboth natural, such as, for example, species-specific or organ-specific,and artificially produced variants of the proteins specificallydescribed herein. Functional equivalents according to the inventiondiffer by addition, substitution, inversion, insertion and/or deletionof one or more amino acid residues of human PARP2 (SEQ ID NO:2), humanPARP3 (SEQ ID NO: 4 and 6) and mouse PARP3 (SEQ ID:8 and 10), therebeing at least retention of the NAD-binding function of the proteinmediated by a functional catalytic C-terminal domain. Likewise, thepoly(ADP-ribose)-producing catalytic activity should preferably beretained. Functional equivalents also comprise where appropriate thosevariants in which the region similar to the leucine zipper isessentially retained.

It is moreover possible, for example, starting from the sequence forhuman PARP2 or human PARP3 to replace certain amino acids by those withsimilar physicochemical properties (bulk, basicity, hydrophobicity,etc.). It is possible, for example, for arginine residues to be replacedby lysine residues, valine residues by isoleucine residues or asparticacid residues by glutamic acid residues. However, it is also possiblefor one or more amino acids to be exchanged in sequence, added ordeleted, or several of these measures can be combined together. Theproteins which have been modified in this way from the human PARP2 orhuman PARP3 sequence have at least 60%, preferably at least 75%, veryparticularly preferably at least 85%, homology with the startingsequence, calculated using the algorithm of Pearson and Lipman, Proc.Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448.

The following homologies have been determined at the amino acid leveland DNA level between human PARP1, 2 and 3 (FastA program, Pearson andLipman, loc. cit.):

Amino acid homologies:

Percent identity Percent identity in PARP signature PARP1/PARP2 41.97%(517)   86% (50) PARP1/PARP3 33.81% (565) 53.1% (49) PARP2/PARP3 35.20%(537) 53.1% (49)

Numbers in parentheses indicate the number of overlapping amino acids.

Percent identity in the ORF Percent identity in PARP signaturePARP1/PARP2 60.81% (467)  77.85% (149) PARP1/PARP3 58.81% (420) 59.02%(61) PARP2/PARP3 60.22% (269) 86.36% (22)

Numbers in parentheses indicate the number of overlapping nucleotides.

The polypeptides according to the invention can be classified ashomologous poly(ADP-ribose) polymerases on the basis of the greatsimilarity in the region of the catalytic domain.

It is also essential to the invention that the novel PARP homologs donot have conventional zinc finger motifs. This means that these enzymesare not necessarily involved in DNA repair or are so in a way whichdiffers from PARP1, but are still able to carry out their pathologicalmechanism (NAD⁺ consumption and thus energy consumption due to ATPconsumption). The strong prote in expression, particularly of PARP3,observable in the Western blot suggests a significant role in the NADconsumption. This is particularly important for drug development.Potential novel inhibitors of the polymerases according to the inventioncan thus inhibit the pathological functions without having adverseeffects on the desired physiological properties. This was impossiblewith inhibitors against the PARPs known to date since there was alwaysalso inhibition of the DNA repair function. The potentially mutageniceffect of known PARP inhibitors is thus easy to understand. It is alsoconceivable to design PARP inhibitors so that they efficiently inhibitall PARP homologs with high affinity. In this case, a potentiated effectis conceivable where appropriate.

The PARP homolog which is preferred according to the invention and isshown in SEQ ID NO:2 (human PARP2) can advantageously be isolated fromhuman brain, heart, skeletal muscle, kidney and liver. The expression ofhuman PARP2 in other tissues or organs is distinctly weaker.

The PARP homolog which is preferred according to the invention and isshown in SEQ ID NO: 4 and 6 (human PARP3) can advantageously be isolatedfrom human brain (in this case very preferentially from thehippocampus), heart, skeletal muscle, liver or kidney. The expression ofhuman PARP3 in other tissues or organs, such as muscle or liver, isdistinctly weaker.

The skilled worker familiar with protein isolation will make use of thecombination of preparative methodologies which is most suitable in eachcase for isolating natural PARPs according to the invention from tissuesor recombinantly prepared PARPs according to the invention from cellcultures. Suitable standard preparative methods are described, forexample, in Cooper, T. G., Biochemische Arbeitsmethoden, published byWalter de Gruyter, Berlin, N.Y. or in Scopes, R. Protein Purification,Springer Verlag, New York, Heidelberg, Berlin.

The invention additionally relates to PARP2 and PARP3 homologs which,although they can be isolated from other eukaryotic species, i.e.invertebrates or vertebrates, especially other mammals such as, forexample, mice, rats, cats, dogs, pigs, sheep, cattle, horses or monkeys,or from other organs such as, for example the myocardium, have theessential structural and functional properties predetermined by thePARPs according to the invention.

In particular, the human PARP2 which can be isolated from human brain,and its functional equivalents, are preferred agents for developinginhibitors of neurodegenerative diseases as for example stroke. This isbecause it can be assumed that drug development based on PARP2 asindicator makes it possible to develop inhibitors which are optimizedfor use in the human brain. However, it cannot be ruled out thatinhibitors developed on the basis of PARP2 can also be employed fortreating PARP-mediated pathological states in other organs, too (seetissue distribution of the proteins according to the invention).

PARP2 and presumably PARP3 are also, similar to PARP1, activated bydamaged DNA, although by a presumably different mechanism.

Significance in DNA repair is conceivable. Blockade of the PARPsaccording to the invention would also be beneficial in indications suchas cancer (e.g. in the radiosensitization of tumor patients).

Another essential biological property of PARPs according to theinvention and their functional equivalents is to be seen in theirability to bind an interacting partner. Human PARP2 and 3 differ frompreviously disclosed PARPs from higher eukaryotes such as, inparticular, mammals by having potential so-called leucine zipper motifs.This is a typical motif for protein-protein interactions. It is possiblethat these motifs permit modulation of PARP activity by an interactingpartner. This additional structural element thus also provides apossible starting point for development of PARP effectors such as, forexample, inhibitors.

The invention thus further relates to proteins which interact with PARP2and/or 3, preferably those which bring about their activation orinactivation.

The invention further relates to proteins which still have theabovementioned ligand-binding activity and which can be preparedstarting from the specifically disclosed amino acid sequences bytargeted modifications.

It is possible, starting from the peptide sequence of the proteinsaccording to the invention, to generate synthetic peptides which areemployed, singly or in combination, as antigens for producing polyclonalor monoclonal antibodies. It is also possible to employ the PARP proteinor fragments thereof for generating antibodies. The invention thus alsorelates to peptide fragments of PARP proteins according to the inventionwhich comprise characteristic partial sequences, in particular thoseoligo- or polypeptides which comprise at least one of the abovementionedsequence motifs. Fragments of this type can be obtained, for example, byproteolytic digestion of PARP proteins or by chemical synthesis ofpeptides.

Novel specific PARP2 and PARP3 binding partners

Active and preferably selective inhibitors against the proteinsaccording to the invention were developed using the specific assaysystems described above for binding partners for PARP2 and PARP3. Theseinhibitors optionally are also active vis a vis PARP1.

Inhibitors provided according to the invention have a strong inhibitoryactivity on PARP2. The K_(i) values may in this case be less than about1000 nM, such as less than about 700 nM, less than about 200 nM or lessthan about 30 nM, e.g. about 1 to 20 nM.

Inhibitors according to the invention may also have a surprisingselectivity for PARP2. This is shown by the K_(i)(PARP1):K_(i)(PARP2)ratio for such inhibitors according to the invention which is, forexample, greater than 3 or greater than 5, as for example greater than10 or greater than 20.

An example which should be mentioned is4-(N-(4-hydroxyphenyl)aminomethyl)-(2H)-dihydrophthalazine-1-one. Thepreparation of this and other analogous compounds may be performedaccording to Puodzhyunas et al., Pharm. Chem. J. 1973, 7, 566 orMazkanowa et al., Zh. Obshch. Khim., 1958, 28, 2798, or Mohamed et al.,Ind. J. Chem. B., 1994, 33, 769 each incorporated by reference.

The above identified compuound shows a K_(i) value of 113 nM for PARP2and is eight times more selective for PARP2 than for PARP3.

Nucleic acids coding for PARP homologs:

Unless stated otherwise, nucleotide sequences are indicated in thepresent description from the 5′ to the 3′ direction.

The invention further relates to nucleic acid sequences which code forthe abovementioned proteins, in particular for those having the aminoacid sequence depicted in SEQ ID NO: 2, 4, 6, 8 and 10, but withoutbeing restricted thereto. Nucleic acid sequences which can be usedaccording to the invention also comprise allelic variants which, asdescribed above for the amino acid sequences, are obtainable bydeletion, inversion, insertion, addition and/or substitution ofnucleotides, preferably of nucleotides shown in SEQ ID NO: 1, 3, 7 and9, but with essential retention of the biological properties and thebiological activity of the corresponding gene product. Nucleotidesequences which can be used are obtained, for example, by nucleotidesubstitutions causing silent (without alteration of the amino acidsequence) or conservative amino acid changes (exchange of amino acids ofthe same size, charge, polarity or solubility).

Nucleic acid sequences according to the invention also embracefunctional equivalents of the genes, such as eukaryotic homologs forexample from invertebrates such as Caenorhabditis or Drosophila, orvertebrates, preferably from the mammals described above. Preferredgenes are those from vertebrates which code for a gene product which hasthe properties essential to the invention as described above.

The nucleic acids according to the invention can be obtained in aconventional way by various routes:

For example, a genomic or a cDNA library can be screened for DNA whichcodes for a PARP molecule or a part thereof. For example, a cDNA libraryobtained from human brain, heart or kidney can be screened with asuitable probe such as, for example, a labeled single-stranded DNAfragment which corresponds to a partial sequence of suitable lengthselected from SEQ ID NO: 0.1 or 3, or sequence complementary thereto.For this purpose, it is possible, for example, for the DNA fragments ofthe library which have been transferred into a suitable cloning vectorto be, after transformation into a bacterium, plated out on agar plates.The clones can then be transferred to nitrocellulose filters and, afterdenaturation of the DNA, hybridized with the labeled probe. Positiveclones are then isolated and characterized.

The DNA coding for PARP homologs according to the invention or partialfragments can also be synthesized chemically starting from the sequenceinformation contained in the present application. For example, it ispossible for this purpose for oligonucleotides with a length of about100 bases to be synthesized and sequentially ligated in a manner knownper se by, for example, providing suitable terminal restriction cleavagesites.

The nucleotide sequences according to the invention can also be preparedwith the aid of the polymerase chain reaction (PCR). For this, a targetDNA such as, for example, DNA from a suitable full-length clone ishybridized with a pair of synthetic oligonucleotide primers which have alength of about 15 bases and which bind to opposite ends of the targetDNA. The sequence section lying between them is then filled in with DNApolymerase.

Repetition of this cycle many times allows the target DNA to beamplified (cf. White et al. (1989), Trends Genet. 5, 185).

The nucleic acid sequences according to the invention are also to beunderstood to include truncated sequences, single-stranded DNA or RNA ofthe coding and noncoding, complementary DNA sequence, mRNA sequences andcDNAs derived therefrom.

The invention further embraces nucleotide sequences hybridizing with theabove sequences under stringent conditions. Stringent hybridizationconditions for the purpose of the present invention exist when thehybridizing sequences have a homology of about 70 to 100%, such as, forexample about 80 to 100% or 90 to 100% (preferably in an amino acidsection of at least about 40, such as, for example, about 50, 100, 150,200, 400 or 500 amino acids).

Stringent conditions for the screening of DNA, in particular cDNA banks,exist, for example, when the hybridization mixture is washed with0.1×SSC buffer (20×SSC buffer=3M NaCl, 0.3M sodium citrate, pH 7.0) and0.1% SDS at a temperature of about 60° C.

Northern blot analyses are analyses are washed under stringentconditions with 0.1×SSC, 0.1% SDS at a temperature of about 65° C., forexample.

Nucleic Acid Derivatives and Expression Constructs:

The nucleic acid sequences are also to be understood to includederivatives such as, for example, promoter variants or alternativesplicing variants. The promoters operatively linked upstream of thenucleotide sequences according to the invention may moreover be modifiedby nucleotide addition(s) or substitution(s), inversion(s), insertion(s)and/or deletion(s), but without impairing the functionality or activityof the promoters. The promoters can also have their activity increasedby modifying their sequence, or be completely replaced by more effectivepromoters even from heterologous organisms. The promoter variantsdescribed above are used to prepare expression cassettes according tothe invention.

Specific examples of human PARP2 splicing variants which may bementioned are:

Variant human PARP2a: Deletion of base pairs 766 to 904 (cf. SEQ IDNO:1). This leads to a frame shift with a new stop codon (“TAA”corresponding to nucleotides 922 to 924 in SEQ ID NO:1).

Variant human PARP2b: Insertion of 5′-gta tgc cag gaa ggt cat ggg ccagca aaa ggg tct ctg-3′ (SEQ ID NO:32) after nucleotide 204 (SEQ IDNO:1). This extends the amino acid sequence by the insertion:GMPGRSWASKRVS (SEQ ID NO:33).

Nucleic acid derivatives also mean variants whose nucleotide sequencesin the region from −1 to −1000 in front of the start codon have beenmodified so that gene expression and/or protein expression is increased.

Besides the nucleotide sequence described above, the nucleic acidconstructs which can be used according to the invention comprise infunctional, operative linkage one or more other regulatory sequences,such as promoters, amplification signals, enhancers, polyadenylationsequences, origins of replication, reporter genes, selectable markergenes and the like. This linkage may, depending on the desired use, leadto an increase or decrease in gene expression.

In addition to the novel regulatory sequences, it is possible for thenatural regulatory sequence still to be present in front of the actualstructural genes. This natural regulation can, where appropriate, beswitched off by genetic modification, and the expression of the genesincreased or decreased. However, the gene construct may also have asimpler structure, that is to say no additional regulatory signals areinserted in front of the structural genes, and the natural promoter withits regulation is not deleted. Instead, the natural regulatory sequenceis mutated in such a way that regulation no longer takes place, and geneexpression is enhanced or diminished. It is also possible to insertadditional advantageous regulatory elements at the 3′ end of the nucleicacid sequences. The nucleic acid sequences can be present in one or morecopies in the gene construct.

Advantageous regulatory sequences for the expression method according tothe invention are, for example, present in promoters such as cos, tac,trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara,SP6, l-PR or the l-PL promoter, which are advantageously used inGram-negative bacteria. Other advantageous regulatory sequences arepresent, for example, in the Gram-positive promoters amy and SPO2, inthe yeast promoters ADC1, MFa, AC, P-60, CYC1, GAPDH or in the plantpromoters CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or in theubiquitin or phaseolin promoter.

It is possible in principle to use all natural promoters with theirregulatory sequences. It is also possible and advantageous to usesynthetic promoters.

Said regulatory sequences are intended to make specific expression ofthe nucleic acid sequences and protein expression possible. This maymean, for example, depending on the host organism that the gene isexpressed or overexpressed only after induction, or that it isimmediately expressed and/or overexpressed.

The regulatory sequences or factors may moreover preferably have apositive influence on, and thus increase or decrease, the expression.Thus, enhancement of the regulatory elements may advantageously takeplace at the level of transcription by using strong transcriptionsignals such as promoters and/or enhancers. However, it is also possibleto enhance translation by, for example, improving the stability of themRNA.

Enhancers mean, for example, DNA sequences which bring about increasedexpression via an improved interaction between RNA polymerase and DNA.

The recombinant nucleic acid construct or gene construct is, forexpression in a suitable host organism, advantageously inserted into ahost-specific vector which makes optimal expression of the genes in thehost possible. Vectors are well known to the skilled worker and are tobe found, for example, in “Cloning Vectors” (Pouwels P. H. et al., Ed.,Elsevier, Amsterdam-N.Y.-Oxford, 1985). Apart from plasmids, vectorsalso mean all other vectors known to the skilled worker, such as, forexample, phages, viruses, such as SV40, CMV, baculovirus and adenovirus,transposons, IS elements, phasmids, cosmids, and linear or circular DNA.These vectors may undergo autonomous replication in the host organism orchromosomal replication.

Expression of the Constructs:

The recombinant constructs according to the invention described aboveare advantageously introduced into a suitable host system and areexpressed. Cloning and transfection methods familiar to the skilledworker are preferably used in order to bring about expression of saidnucleic acids in the particular expression system. Suitable systems aredescribed, for example, in Current Protocols in Molecular Biology, F.Ausubel et al., ed., Wiley Interscience, New York 1997.

Suitable host organisms are in principle all organisms which make itpossible to express the nucleic acids according to the invention, theirallelic variants, their functional equivalents or derivatives or therecombinant nucleic acid construct. Host organisms mean, for example,bacteria, fungi, yeasts, plant or animal cells. Preferred organisms arebacteria such as those of the genera Escherichia, such as, for example,Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryoticmicroorganisms such as Saccharomyces cerevisiae, Aspergillus, highereukaryotic cells from animals or plants, for example Sf9 or CHO cells.

The gene product can also, if required, be expressed in transgenicorganisms such as transgenic animals such as, in particular, mice,sheep, or transgenic plants. The transgenic organisms may also beso-called knock-out animals or plants in which the correspondingendogenous gene has been switched off, such as, for example, by mutationor partial or complete deletion.

The combination of the host organisms and the vectors appropriate forthe organisms, such as plasmids, viruses or phages, such as, forexample, plasmids with the RNA polymerase/promoter system, phages λ, μor other temperate phages or transposons and/or other advantageousregulatory sequences forms an expression system. The term expressionsystems preferably means, for example, a combination of mammalian cellssuch as CHO cells, and vectors, such as pcDNA3neo vector, which aresuitable for mammalian cells.

As described above, the gene product can also be expressedadvantageously in transgenic animals, e.g. mice, sheep, or transgenicplants. It is likewise possible to program cell-free translation systemswith the RNA derived from the nucleic acid.

The gene product can also be expressed in the form of therapeutically ordiagnostically suitable fragments. To isolate the recombinant protein itis possible and advantageous to use vector systems or oligonucleotideswhich extend the cDNA by certain nucleotide sequences and thus code formodified polypeptides which serve to simplify purification. Suitablemodifications of this type are, for example, so-called tags which act asanchors, such as, for example, the modification known as thehexa-histidine anchor, or epitopes which can be recognized as antigensby antibodies (described, for example, in Harlow, E. and Lane, D., 1988,Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Theseanchors can be used to attach the proteins to a solid support such as,for example, a polymer matrix, which can, for example, be packed into achromatography column, or to a microtiter plate or to another support.

These anchors can also at the same time be used to recognize theproteins. It is also possible to use for recognition of the proteinsconventional markers such as fluorescent dyes, enzyme markers which forma detectable reaction product after reaction with a substrate, orradioactive markers, alone or in combination with the anchors forderivatizing the proteins.

Production of Antibodies:

Anti-PARP2 antibodies are produced in a manner familiar to the skilledworker. Antibodies mean both polyclonal, monoclonal, human or humanizedantibodies or fragments thereof, single chain antibodies or alsosynthetic antibodies, likewise antibody fragments such as Fv, Fab andF(ab′)₂. Suitable production methods are described, for example, inCampbell, A. M., Monoclonal Anti-body Technology, (1987) ElsevierVerlag, Amsterdam, N.Y., Oxford and in Breitling, F. and Dübel, S.,Rekombinante Antikörper (1997), Spektrum Akademischer Verlag,Heidelberg.

Further Use of the Coding Sequence:

The present cDNA additionally provides the basis for cloning the genomicsequence of the novel PARP genes. This also includes the relevantregulatory or promoter sequence, which is available, for example, bysequencing the region located 5′ upstream of the cDNA according to theinvention or located in the introns of the genes. The cDNA sequenceinformation is also the basis for producing antisense molecules orribozymes with the aid of known methods (cf. Jones, J. T. and Sallenger,B. A. (1997) Nat. Biotechnol. 15, 902; Nellen, W. and Lichtenstein, C.(1993) TIBS, 18, 419). The genomic DNA can likewise be used to producethe gene constructs described above.

Another possibility of using the nucleotide sequence or parts thereof isto generate transgenic animals. Transgenic overexpression or geneticknock-out of the sequence information in suitable animal models mayprovide further valuable information about the (patho)physiology of thenovel genes.

Therapeutic Applications:

In situations where there is a prevailing deficiency of a proteinaccording to the invention it is possible to employ several methods forreplacement. On the one hand, the protein, natural or recombinant, canbe administered directly or by gene therapy in the form of its codingnucleic acid (DNA or RNA). It is possible to use any suitable vectorsfor this, for example both viral and non-viral vehicles. Suitablemethods are described, for example, by Strauss and Barranger in Conceptsin Gene Therapy (1997), Walter de Gruyter, publisher. Anotheralternative is provided by stimulation of the endogenous gene bysuitable agents.

It is also possible to block the turnover or the inactivation of PARPsaccording to the invention, for example by proteases. Finally,inhibitors or agonists of PARPs according to the invention can beemployed.

In situations where a PARP is present in excess or is overactivated,various types of inhibitors can be employed. This inhibition can beachieved both by antisense molecules, ribozymes, oligonucleotides orantibodies, and by low molecular weight compounds.

The active substances according to the invention, i.e. PARP proteins,nucleic acids and PARP binding partners such as, for example, antibodiesor modulators, can be administered either as single therapeutic activesubstances or as mixtures with other therapeutic active substances. Theycan be administered as such, but in general they are administered in theform of pharmaceutical compositions, i.e. as mixtures of the activesubstance(s) with at least one suitable pharmaceutical carrier ordiluent. The active substances or compositions can be administered inany way suitable for the particular therapeutic purpose, e.g. orally orparenterally.

The nature of the pharmaceutical composition and of the pharmaceuticalcarrier or diluent depends on the required mode of administration. Oralcompositions can be, for example, in the form of tablets or capsules andmay contain customary excipients such as binders (e.g. sirup, acacia,gelatin, sorbitol, tragacanth or polyvinylpyrrolidone), bulking agents(e.g. lactose, sugar, corn starch, calcium phosphate, sorbitol orglycine), lubricants (e.g. magnesium stearate, talc, polyethylene glycolor silica), disintegrants (e.g. starch) or wetting agents (e.g. sodiumlauryl sulfate). Oral liquid products may be in the form of aqueous oroily suspensions, solutions, emulsions, sirups, elixirs or sprays etc.or may be in the form of dry powders for reconstitution with water oranother suitable carrier. Liquid products of these types may containconventional additives, for example suspending agents, flavorings,diluents or emulsifiers. It is possible to employ for parenteraladministration solutions or suspensions with conventional pharmaceuticalcarriers. Parenteral administration of active substances according tothe invention advantageously takes place using a liquid pharmaceuticalcomposition which can be administered parenterally, in particularintravenously. This preferably contains an effective amount of at leastone active substance, preferably in dissolved form, in apharmaceutically acceptable carrier suitable for this purpose. Examplesof pharmaceutical carriers suitable for this purpose are, in particular,aqueous solutions such as, for example, physiological saline,phosphate-buffered saline, Ringer's solution, Ringer's lactate solutionand the like. The composition may moreover contain further additionssuch as antioxidants, chelating agents or antimicrobial agents.

The choice in each case of the dosage of the active substances accordingto the invention and the particular dosage schedule are subject to adecision of the treating physician. The latter will select a suitabledose and an appropriate dosage schedule depending on the chosen route ofadministration, on the efficacy of the medicine in each case, on thenature and severity of the disorder to be treated, and on the conditionof the patient and his response to the therapy. Thus, for example, thepharmacologically active substances can be administered to a mammal(human or animal) in doses of about 0.5 mg to about 100 mg per kg ofbody weight and day. They can be administered in a single dose or inseveral doses.

Nontherapeutic Applications:

The nucleic acids according to the invention, such as, for example,cDNA, the genomic DNA, the promoter, and the polypeptide, and partialfragments thereof, can also be used in recombinant or nonrecombinantform for developing various test systems.

For example, it is possible to establish a test system which is suitablefor measuring the activity of the promoter or of the protein in thepresence of a test substance. The methods of measurement in this caseare preferably simple ones, e.g. colorimetric, luminometric,fluorimetric, immunological or radioactive, and allow preferably a largenumber of test substances to be measured rapidly. Tests of this type aresuitable and advantageous for so-called high-throughput screening. Thesetest systems allow test substances to be assessed for their binding toor their agonism, antagonism or inhibition of proteins according to theinvention.

Determination of the amount, activity and distribution of the proteinsaccording to the invention or their underlying mRNA in the human bodycan be used for the diagnosis, for the determination of thepredisposition and for the monitoring of certain diseases. Likewise, thesequence of the cDNA and the genomic sequence may provide informationabout genetic causes of and predispositions to certain diseases. It ispossible to use for this purpose both DNA/RNA probes and antibodies of awide variety of types. The nucleotide sequences according to theinvention or parts thereof can further be used in the form of suitableprobes for detecting point mutations, deletions or insertions.

The proteins according to the invention can further be used to identifyand isolate their natural ligands or interacting partners. The proteinsaccording to the invention can additionally be used to identify andisolate artificial or synthetic ligands. For this purpose, therecombinantly prepared or purified natural protein can be derivatized insuch a way that it has modifications which permit linkage to supportmaterials. Proteins bound in this way can be incubated with variousanalytes, such as, for example, protein extracts or peptide libraries orother sources of ligands. Specifically bound peptides, proteins or lowmolecular weight, non-proteinogenous substances can be isolated andcharacterized in this way. Non-proteinogenous substances mean, forexample, low molecular weight chemical substances which may originate,for example, from classical drug synthesis or from so-called substancelibraries which have been synthesized combinatorially.

The protein extracts used are derived, for example, from homogenates ofplants or parts of plants, microorganisms, human or animal tissues ororgans.

Ligands or interacting partners can also be identified by methods likethe yeast two-hybrid system (Fields, S, and Song, O. (1989) Nature, 340,245). The expression banks which can be employed in this case may bederived, for example, from human tissues such as, for example, brain,heart, kidney etc.

The nucleic acid sequences according to the invention and the proteinsencoded by them can be employed for developing reagents, agonists andantagonists or inhibitors for the diagnosis and therapy of chronic andacute diseases associated with the expression or activation of one ofthe protein sequences according to the invention, such as, for example,with increased or decreased expression thereof. The reagents, agonists,antagonists or inhibitors developed can subsequently be used to producepharmaceutical preparations for the treatment or diagnosis of disorders.Examples of possible diseases in this connection are those of the brain,of the peripheral nervous system, of the cardiovascular system or of theeye, of septic shock, of rheumatoid arthritis, diabetes, acute kidneyfailure, or of cancer.

The relevance of the proteins according to the invention for saidindications was verified using specific inhibitors in relevant animalmodels.

The invention is now illustrated in detail with reference to thefollowing examples.

Example 1 Isolation of the PARP2 and PARP3 cDNA

The present cDNA sequences were found for the first time on sequenceanalysis of cDNA clones of a cDNA library from human brain (Human Brain5′Stretch Plus cDNA Library, # HL3002a, Clontech). The mouse PARP3clones were isolated from a “lambda triplex mouse brain cDNA library”(Clontech order No. ML5004t). The sequences of these clones aredescribed in SEQ ID NO:1, 3, 7 and 9.

Example 2 Expression of PARP2 and PARP3 in Human Tissues

The expression of human PARP2 and human PARP3 was investigated in twelvedifferent human tissues by Northern blot analysis. A Human MultipleTissue Northern Blot (MTN™) supplied by Clontech (#7760-1 and #7780-1)was hybridized for this purpose with an RNA probe. The probe wasproduced by in vitro transcription of the corresponding cDNA of humanPARP2 and human PARP3 in the presence of digoxigenin-labeled nucleotidesin accordance with the manufacturer's method (BOEHRINGER MANNHEIM DIGEasy Hyb order No. 1603 558, DIG Easy Hyb method for RNA:RNAhybridization). The protocol was modified to carry out theprehybridization: 2×1 h with addition of herring sperm DNA (10 mg/ml ofhybridization solution). Hybridization then took place overnight withaddition of herring sperm DNA (10 mg/ml of hybridization solution). Thebands were detected using the CDP-Star protocol (BOEHRINGER MANNHEIMCDP-Star™ order No. 1685 627).

After stringent washing, the transcript of PARP2 was mainly detected inhuman brain, heart, skeletal muscle, kidney and liver. The transcriptsize of about 1.9 kb corresponds to the length of the cDNA determined(1.85 kb) (cf. FIG. 2(A)).

In other tissues or organs, human PARP2 expression is considerablyweaker.

After stringent washing, the transcript of PARP3 was mainly detected inheart, brain, kidney, skeletal muscle and liver. Expression in othertissues (placenta, lung, pancreas) is distinctly weaker (cf. FIG. 2(B)).There are at least 2 transcripts for human PARP3, which can presumablybe explained by different polyadenylation sites or alternative splicing.Their size (about 2.2 kb and 2.5 kb respectively) corresponds to thelength of the cDNA determined (2.3 kb). Washing was carried out with0.2×SSC/0.2% SDS at room temperature for 2×15 minutes and then with0.1×SSC/0.1% SDS at 65° C. for 2×15 minutes (prepared from 20×SSC: 3MNaCl, 0.3M sodium citrate, pH 7.0).

Example 3 Production of Antibodies

Specific antibodies against the proteins according to the invention wereproduced. These were used inter alia for analyzing the tissuedistribution at the protein level of PARP2 and PARP3 by immunoblot(Western blot) analysis. Examples of the production of such antibodiesare indicated below.

The following peptides were prepared by synthesis in the manner familiarto the skilled worker for the antibody production. In some cases, acysteine residue was attached to the N or C terminals of the sequencesin order to facilitate coupling to KLH (keyhole limpet hemocyanin).

-   PARP-2: NH₂-MAARRRRSTGGGRARALNES-CO₂H (amino acids 1-20; SEQ ID NO:    23)-    NH₂-KTELQSPEHPLDQHYRNLHC—CO₂H (amino acids 335-353; SEQ ID NO: 24)-    PARP-3: NH₂-CKGRQAGREEDPFRSTAEALK-CO₂H (amino acids 25-44 SEQ ID    NO: 25)-    NH₂-CKQQIARGFEALEALEEALK-CO₂H (amino acids 230-248; SEQ ID NO: 26)

The production of an anti-PARP3 antibody is described as arepresentative example.

For human PARP3, polyclonal antibodies were raised in rabbits using asynthetic peptide having the peptide sequenceH₂N-KQQIARGFEALEALEEALK-CO₂H (SEQ ID NO: 27) (amino acids 230-248 of thehuman PARP3 protein sequence). The corresponding mouse sequence differsin this region only by one amino acid (H₂N-KQQIARGFEALEALEEAMKCO₂H; SEQID NO: 28). A cysteine was also attached to the N terminus in order tomake it possible for the protein to couple to KLH.

Rabbits were immunized a total of five times, at intervals of 7-14 days,with the KLH-peptide conjugate. The antiserum obtained wasaffinity-purified using the antigen. The specific IgG fraction wasisolated from the serum using the respective peptides which, for thispurpose, were initially immobilized on an affinity column in the mannerfamiliar to the skilled worker. The respective antiserum was loaded ontothis affinity column, and non-specifically sorbed proteins were elutedwith buffer. The specifically bound IgG fraction was eluted with 0.2 Mglycine/HCl buffer pH 2.2. The pH was immediately increased using a 1MTRIS/HCl buffer pH 7.5. The eluate containing the IgG fraction was mixed1:1 (volume) with saturated ammonium sulfate solution and incubated at+4° C. for 30 min to complete the precipitation. The resultingprecipitate was centrifuged at 10,000 g and, after removal of thesupernatant, dissolved in the minimum amount of PBS/TBS. The resultingsolution was then dialyzed against PBS/TBS in the ratio 1:100 (volume).The antibodies were adjusted to a concentration of about 100 μg ofIgG/ml. The PARP3 antibodies purified in this way had high specificityfor PARP3. Whereas mouse PARP3 was recognized well, there was noobservable cross-reaction with PARP1 or PARP2.

Example 4 Analysis of the Tissue Distribution by Immunoblot (WesternBlot)

The tissue distribution at the protein level was also investigated forPARP2 and PARP3 by immunoblot (Western blot) analysis.

Preparation of the Mouse Tissues for Protein Gels:

Tissues or cells were homogenized using a Potter or Ultra-Turrax. Forthis, 0.5 g of tissue (or cells) was incubated in 5 ml of buffer (10 mMTris-HCl pH 7.5, 1 mM EDTA, 6 mM MgCl₂), one tablet of proteaseinhibitor cocktail (Boehringer Mannheim, order No.: 1836153) andbenzonase (purity grade I, MERCK) at 37° C. for 30 min. Tissue samplesfrom mice were produced for heart, lung, liver, spleen, kidney,intestine, muscle, brain and for human embryonic kidney cells (HEK293,human embryonal kidney).

Protein Gels:

The NuPAGE system supplied by NOVEX was used according to theinstructions for protein gels. Polyacrylamide gels (NuPAGE 4-12%BisTris, NOVEX NP 0321), running buffer (MES-Running Buffer, NOVEX NP0002), antioxidant (NOVEX NP 0005), protein size standard (Multi MarkMulti Colored Standard, NOVEX LC 5725), sample buffer (NuPAGE LDS SampleBuffer (4×), NOVEX NP 0007) were used. The gels were run for 45 minutesat a voltage of 200 V.

Western Blot:

Western blots were carried out using the NOVEX system in accordance withinstructions. A nitrocellulose membrane (Nitrocellulose Pore size 45 μm,NOVEX LC 2001) was used. The transfer took 1 hour at a current of 200mA. The transfer buffer consisted of 50 ml of transfer bufferconcentrate (NOVEX NP 0006), 1 ml of antioxidant (NOVEX NP 0002), 100 mlof analytical grade methanol and 849 ml of double-distilled water.

Besides the blots produced in this way, also used were premade blots,for example from Chemicon (mouse brain blot, Chemicon, catalog No.: NS106 with the tissues 1. frontal cortex, 2. posterior cortex, 3.cerebellum, 4. hippocampus, 5. olfactory bulb, 6. striatum, 7. thalamus,8. mid brain, 9. entorhinal cortex, 10. pons, 11. medulla, 12. spinalcord).

Antibody Reaction with PARP3:

The Western blots were blocked in TBST (TBS+0.3% Tween 20) with 5% drymilk powder for at least 2 hours (TBS: 100 mM Tris pH 7.5, 200 mM NaCl).The antibody reaction with the primary anti-body (dilution 1:1000) tookplace in TBST with 5% dry milk powder (see above) at room temperaturefor at least 2 hours or at 4° C. overnight, with gentle agitation(vertical rotator). This was followed by washing three times in TBST for5 minutes. Incubation with the secondary antibody (anti-rabbit IgG,peroxidase-coupled, SIGMA A-6154, dilution 1:2000) took place in TBSTwith 5% dry milk powder for 1 hour. This was followed by washing threetimes for 5 minutes each time as above. The subsequent detection wasbased on chemiluminescence using the SUPER BLAZE kit (Pierce, SignalBLAZE Chemiluminescent Substrate 34095) as stated by the manufacturer.The “Lumi-Film” (Chemiluminescent Detection Film, Boehringer order No:1666916) was used. The films were developed for about 2 min (X-raydeveloper concentrate, ADEFO-Chemie GmbH), hydrated, fixed for about 4min (Acidofix 85 g/l/AGFA), hydrated and then dried.

Example 5 Preparation of the Enzymes

For comparison, human PARP1 was expressed recombinantly in thebaculovirus system in the manner familiar to the skilled worker andpartially purified as described (Shah et al., Analytical Biochemistry1995, 227, 1-13). Bovine PARP1 in a purity of 30-50% (c=0.22 mg/ml,spec. activity 170 nmol of ADP-ribose/min/mg of total protein at 25° C.)was purchased from BIOMOL (order No. SE-165). Human and mouse PARP2 andPARP3 were expressed recombinantly in the baculovirus system (Bac-to-Bacsystem, BRL LifeScience). For this purpose, the appropriate cDNAs werecloned to the pFASTBAC-1 vector. Preparation of recombinant baculovirusDNA by recombination in E. coli was followed by transfection of insectcells (Sf9 or High-Five) with the appropriate recombinant baculovirusDNAs. Expression of the corresponding proteins was verified by Westernblot analysis. Virus strains were amplified in the manner familiar tothe skilled worker. Larger amounts of recombinant proteins were obtainedby infecting 500 ml of insect cell culture (2×10⁶ cells/ml) with virusesin an MOI (multiplicity of infection; ratio of viruses to cells) of 5-10and incubating for 3 to 4 days. The insect cells were then pelleted bycentrifugation, and the proteins were purified from the pellet.

The purification took place by classical methods of protein purificationfamiliar to the skilled worker, detecting the enzymes with appropriatespecific antibodies. In some cases, the proteins were alsoaffinity-purified on a 3-aminobenzamide affinity column as described(Burtscher et al., Anal Biochem 1986, 152:285-290). The purity was >90%.

Example 6 Assay Systems for Determining the Activity of PARP2 and PARP3and the Inhibitory Action of Effectors on PARP1, PARP2 and PARP3

a) Production of Antibodies Against poly(ADP-ribose)

It is possible to use poly(ADP-ribose) as antigen for generatinganti-poly(ADP-ribose) antibodies. The production of antipoly(ADP-ribose)antibodies is described in the literature (Kanai Y et al. (1974) BiochemBiophys Res Comm 59:1, 300-306; Kawamaitsu H et al. (1984) Biochemistry23, 3771-3777; Kanai Y et al. (1978) Immunology 34, 501-508).

The following were used, inter alia: anti-poly(ADP-ribose) antibodies(polyclonal antiserum, rabbits), BIOMOL; order No. SA-276,anti-poly(ADP-ribose) antibodies (monoclonal, mouse; clone 10H;hybridoma supernatant, affinity-purified).

The antisera or monoclonal antibodies obtained from hybridomasupernatant were purified by protein A affinity chromatography in themanner familiar to the skilled worker.

b) ELISA

Materials:

ELISA Color Reagent: TMB Mix, SIGMA T-8540

A 96-well microtiter plate (FALCON Micro-Test Flexible Assay Plate, #3912) was coated with histones (SIGMA, H-7755). Histones were for thispurpose dissolved in carbonate buffer (0.05M Na₂HCO₃; pH 9.4) in aconcentration of 50 μg/ml. The individual wells of the microtiter platewere each incubated with 150 μl of this histone solution at roomtemperature for at least 2 hours or at 4° C. overnight. The wells arethen blocked by adding 150 μl of a 1% BSA solution (SIGMA, A-7888) incarbonate buffer at room temperature for 2 hours. This is followed bythree washing steps with washing buffer (0.05% Tween10 in 1×PBS; PBS(Phosphate buffered saline; Gibco, order No. 10010): 0.21 g/l KH₂PO₄, 9g/l NaCl, 0.726 g/l Na₂HPO₄.7H₂O, pH 7.4). Washing steps were allcarried out in a microtiter plate washer (“Columbus” microtiter platewasher, SLT-Labinstruments, Austria).

Required for the enzyme reaction were an enzyme reaction solution and asubstrate solution, in each case as a premix. The absolute amount ofthese solutions depended on the intended number of assay wells.

Composition of the enzyme reaction solution per well:

-   -   4 μl of PARP reaction buffer (1M Tris-HCl pH 8.0, 100 mM MgCl₂,        10 mM DTT)    -   20 ng of PARP1 (human or bovine) or 8 ng PARP2 (human or mouse)    -   4 μl of activated DNA (1 mg/ml; SIGMA, D-4522)    -   H₂O ad 40 μl

Composition of the substrate solution per well:

-   -   5 μl of PARP reaction buffer (10×)    -   0.8 μl of NAD solution (10 mM, SIGMA N-1511)    -   44 μl H₂O

Inhibitors were dissolved in 1× PARP reaction buffer. DMSO, which wasoccasionally used to dissolve inhibitors in higher concentrations, wasno problem up to a final concentration of 2%. For the enzyme reaction,40 μl of the enzyme reaction solution were introduced into each well andincubated with 10 μl of inhibitor solution for 10 minutes. The enzymereaction was then started by adding 50 μl of substrate solution perwell. The reaction was carried out at room temperature for 30 minutesand then stopped by washing three times with washing buffer.

The primary antibodies employed were specific anti-poly(ADP-ri-Bose)antibodies in a dilution of 1:5000. Dilution took place in antibodybuffer (1% BSA in PBS; 0.05% Tween20). The incubation time for theprimary antibodies was one hour at room temperature. After subsequentlywashing three times with washing buffer, incubation was carried out withthe secondary antibody (anti-mouse IgG, Fab fragments,peroxidase-coupled, Boehringer Mannheim, order No. 1500.686; anti-rabbitIgG, peroxidase-coupled, SIGMA, order No. A-6154) in a dilution of1:10,000 in antibody buffer at room temperature for one hour. Washingthree times with washing buffer was followed by the color reaction using100 μl of color reagent (TMB mix, SIGMA) per well at room temperaturefor about 15 min. The color reaction was stopped by adding 100 μl of 2MH₂SO₄. This was followed by immediate measurement in an ELISA platereader (EAR340AT “Easy Reader”, SLT-Labinstruments, Austria) (450 nmversus 620 nm). The measurement principle is depicted diagrammaticallyin FIG. 6.

Various concentrations were used to construct a dose-effect plot todetermine the K_(i) value of an inhibitor. Values are obtained intriplicate for a particular inhibitor concentration. Arithmetic meansare determined using Microsoft© Excel. The IC₅₀ is determined using theMicrocal© Origin Software (Vers. 5.0) (“Sigmoidal Fit”). Conversion ofthe IC₅₀ value is calculated in this way into K_(i) values took place byusing “calibration inhibitors”. The “calibration inhibitors” were alsomeasured in each analysis. The K_(i) values of the “calibrationinhibitors” were determined in the same assay system by analysis of theDixon diagram in the manner familiar to the skilled worker.

b) HTRF (Homogenous Time-Resolved Fluorescence) Assay

In the HTRF PARP assay according to the invention, histones, as targetproteins for modification by PARP, are labeled indirectly with an XL665fluorophore. The anti poly(ADP ribose) antibody is directly labeled witha europium cryptate (anti-PAR-cryptate). If the XL665 fluorophore is inthe direct vicinity in space, which is ensured by binding to thepoly(ADP-ribose) on the histone, then energy transfer is possible. Theemission at 665 nm is thus directly proportional to the amount of boundantibody, which in turn is equivalent to the amount of poly(ADP-ribose).The measured signal thus corresponds to the PARP activity. Themeasurement principle is depicted diagrammatically in FIG. 7. Thematerials used are identical to those used in the ELISA (see above)unless expressly indicated.

Histones were dissolved in a concentration of 3 mg/ml in Hepes buffer(50 mM, pH=7.5). Biotinylation took place with sulfo-NHS-LC-biotin(Pierce, #21335T). A molar ratio of 4 biotin molecules per histone wasused. The incubation time was 90 minutes (RT). The biotinylated histoneswere then purified on a G25 SF HR10/10 column (Pharmacia, 17-0591-01) inHepes buffer (50 mM, pH=7.0) in order to remove excess biotinylationreagent. The anti-poly(ADP-ribose) antibody was labeled with europiumcryptate using bifunctional coupling reagents (Lopez, E. et al., Clin.Chem. 39(2), 196-201 (1993); U.S. Pat. No. 5,534,622).

Purification took place on a G25SF HR10/30 column. A molar ratio of 3.1cryptates per antibody was achieved. The yield was 25%. The conjugateswere stored at −80° C. in the presence of 0.1% BSA in phosphate buffer(0.1M, pH=7).

For the enzyme reaction, the following were pipetted into each well:

-   -   10 μl of PARP solution in PARP HTRF reaction buffer (50 mM        TrisHCl pH 8.0, 10 mM MgCl₂, 1 mM DTT) with 20 ng of PARP1        (human or bovine) or 8 ng of PARP2 (human or mouse)    -   10 μl of activated DNA in PARP HTRF reaction buffer (50 μg/ml)    -   10 μl of biotinylated histones in PARP HTRF reaction buffer        (1.25 μM)    -   10 μl of inhibitor in PARP HTRF reaction buffer

These reagents were incubated for 2 minutes before the reaction wasstarted by adding

-   -   10 μl of NAD solution in PARP HTRF reaction buffer (41 μM/ml).

The reaction time was 30 minutes at room temperature.

The reaction was then stopped by adding

-   -   10 μl of PARP inhibitor (25 μM, K_(i)=10 nM) in “Revelation”        buffer (100 mM Tris-HCl pH 7.2, 0.2M KF, 0.05% BSA).

The following were then added:

-   -   10 μl of EDTA solution (SIGMA, E-7889, 0.5M in H₂O)    -   100 μl of Sa-XL665 (Packard Instruments) in “Revelation” buffer        (15-31.25 nM)    -   50 μl of anti-PAR cryptate in “Revelation” buffer (1.6-3.3 nM).

Measurement was then possible after 30 minutes (up to 4 hours). Themeasurement took place in a “discovery HTRF microplate analyzer” (Canberra Packard Instruments). The K_(i) values were calculated asdescribed for the ELISA.

Example 7 Test Systems for Determining the Therapeutic Efficacy of PARPInhibitors

Novel PARP inhibitors can have their therapeutic efficacy checked inrelevant pharmacological models. Examples of some suitable models arelisted in Table 1.

Disorder Model Literature Neurodegenerative NMDA excitotoxicity Seebelow for disorders (stroke, in mice or rats description Parkinson's,etc.) Stroke Permanent MCAO Tokime, T. et al., (“middle cerebral J.Cereb. Blood Flow arterial occlusion”) Metab., 18(9): 991-7, 1998.Guegan, C., Brain Research. Molecular Brain Research, 55(1): 133-40,1998. Transient, focal Eliasson MJL et al., MCAO in rats or mice Nat Med1997, 3:1089-1095. Endres, M et al., J Cereb Blood Flow Metab 1997, 17:1143-1151. Takahashi K et al., J Cereb Blood Flow Metab 1997, 17:1137-1142. Parkinson's disease MPTP (1-methyl- Cosi C, et al.,4-phenyl-1,2,3,6- Brain Res. ,1998 tetrahydropyridine) 809(1): 58-67.toxicity in mice/ Cosi C, et al. rats Brain Res., 1996 729(2): 264-9.Myocardial infarct Coronary vessel Richard V, et al., occlusion in rats,Br. J. Pharmacol pigs or rabbits 1994, 113, 869-876. Thiemermann C, etal., Proc Natl Acad Sci U S A. 1997, 94(2): 679-83. Zingarelli B, etal., Cardiovasc Res. 1997, 36(2): 205-15. Langendorf heart See below formodel in rats or description rabbits Septic shock Endotoxin shock inSzabo C, et al., J rats Clin Invest, 1997, 100(3): 723-35. Zymosan- orSzabo C, et al. J carrageenan-induced Exp Med. 1997, multiple organ186(7): 1041-9. failure in rats or Cuzzocrea S, et al. Eur J micePharmacol. 1998, 342(1): 67-76. Rheumatoid arthritis Adjuvant- or SzaboC, et al., collagen-induced Proc Natl Acad Sci arthritis in rats or U SA. 1998, mice 95(7): 3867-72. Diabetes Streptozotocin- and Uchgata Y etal., alloxan-induced or Diabetes 1983, 32: obesity-associated 316-318.Masiello P et al., Diabetologia 1985, 28: 683-686. Shimabukuro M et al.,J Clin Invest 1997, 100: 290-295. Cancer In vitro model; Schlicker etal., see below 1999, 75(1), 91-100.a) NMDA Excitotoxicity Model

Glutamate is the most important excitory neurotransmitter in the brain.Under normal conditions, glutamate is secreted into the synaptic cleftand stimulates the post-synaptic glutamate receptors, specifically theglutamate receptors of the “NMDA” and “AMPA” types. This stimulationplays a significant part in numerous functions of the brain, includinglearning, memory and motor control.

Under the conditions of acute and chronic neurodegeneration (e.g.stroke), however, there is a great increase in the presynaptic glutamatesecretion, resulting in excessive stimulation of the receptors. Thisleads to death of the cells stimulated in this way. These increasedglutamate activities occur in a number of neurological disorders orpsychological disturbances and lead to states of overexcitation or toxiceffects in the central nervous system (CNS) but also in the peripheralnervous system. Thus, glutamate is involved in a large number ofneurodegenerative disorders, in particular neurotoxic disturbancesfollowing hypoxia, anoxia, ischemia and after lesions like thoseoccurring after stroke and trauma, and stroke, Alzheimer's disease,Huntington's disease, amyotrophic lateral sclerosis (ALS; “Lou Gehring'sdisease”), cranial trauma, spinal cord trauma, peripheral neuropathies,AIDS dementia and Parkinson's disease. Another disease in whichglutamate receptors are important is epilepsy (cf. Brain Res Bull 1998;46(4):281-309, Eur Neuropsychopharmacol 1998, 8(2):141-52.).

Glutamate effects are mediated through various receptors. One of thesereceptors is called the NMDA (N-methyl-D-aspartate) receptor after aspecific agonist (Arzneim.Forschung 1990, 40, 511-514; TIPS, 1990, 11,334-338; Drugs of the Future 1989, 14, 1059-1071). N-Methyl-D-aspartateis a strong agonist of a particular class of glutamate receptors (“NMDA”type). Stimulation of the NMDA receptor leads to influx of calcium intothe cell and the generation of free radicals. The free radicals lead toDNA damage and activation of PARP. PARP in turn causes cell deaththrough depletion of high-energy phosphates (NAD and ATP) in the cell.This explains the toxicity of NMDA. Treatment of animals with NMDA cantherefore be regarded as a model of the abovementioned disorders inwhich excitotoxicity is involved.

Because of the importance of glutamate receptors in neurodegeneration,many pharmacological approaches to date have been directed at specificblocking of precisely these receptors. However, because of theirimportance in normal stimulus conduction, these approaches have provedto be problematic (side effects). In addition, stimulation of thereceptors is an event which takes place very rapidly so thatadministration of the receptors often comes too late (“time window”problem). Thus there is a great need for novel principles of action andinhibitors of NMDA-related neurotoxicity.

Protection against cerebral overexcitation by excitatory amino acids(NMDA antagonism in mice) can be regarded as adequate proof of theactivity of a pharmacological effector of PARP in disorders based onexcitotoxicity. Intracerebral administration of excitatory amino acids(EAA) induces such massive overexcitation that it leads within a shorttime to convulsions and death of the animals (mice).

In the present case there was unilateral intracerebroventricularadministration of 10 μl of a 0.035% strength aqueous NMDA solution 120minutes after intraperitoneal (i.p.) administration of the testsubstance. These symptoms can be inhibited by systemic, e.g.intraperitoneal, administration of centrally acting drugs. Sinceexcessive activation of EAA receptors in the central nervous systemplays an important part in the pathogenesis of various neurologicaldisorders, information can be gained from the detected EAA antagonism invivo about possible therapeutic utilizability of the substances for suchCNS disorders. An ED50 at which 50% of the animals are, due to precedingi.p. administration of the measured substance, free of symptoms with afixed dose of NMDA was determined as a measure of the activity of thesubstances.

b) Langendorf Heart Model (Model for Myocardial Infarct)

Male Sprague-Dawley rats (bodyweight 300-400 g; origin Janvier, LeGenest-St-Isle, France) were used for the test. The rats were treatedorally by gavage with the active substance or placebo (volume: 5 ml/kg).50 minutes later, heparin is administered intraperitoneally (Liquemin NRoche, 125 IU/animal in 0.5 ml). The animals are anesthesized withInactin® T133 (thiobetabarbital sodium 10%), fixed on the operatingtable, tracheotomized and ventilated with a “Harvard ventilatory pump”(40 beats/min, 4.5 ml/beat). Thoracotomy was followed by immediatecatheterization of the aorta, removal of the heart and immediateretrograde perfusion. The hearts were perfused with a constant pressureof 75 mmHg, which is achieved using a “Gilson Miniplus 2 perfusionpump”. Composition of the perfusate (mmol/l): NaCl 118, KCl 4.7,CaCl₂×2H₂O 2.52, MgSO₄×7H₂O 1.64, NaHCO₃ 24.88, KH₂PO₄ 1.18, glucose 11.The temperature is kept at 37° C. throughout the experiment. Functionalparameters were continuously recorded using a “Gould 4-channelrecorder”. Measurements were made of the left-ventricular pressure (LVP;mmHg), LVEDP (mmHg), enzyme release (creatine kinase, mU/ml/g), coronaryflow rate (ml/min), HR (pulse rate, min⁻¹). The left-ventricularpressure was measured using a liquid-filled latex balloon and aStatham23 Db pressure transducer. The volume of the balloon wasinitially adjusted to reach an LVEDP (left-ventricular end-diastolicpressure) of about 12 mmHg. The dP/dt_(max) (maximum pumping force) isderived from the pressure signal using a differentiator module. Theheart rate was calculated from the pressure signal. The flow rate wasdetermined using a drop counter (BMT Messtechnik GmbH Berlin). After anequilibration time of 20 minutes, the hearts were subjected to a30-minute global ischemia by stopping the perfusate supply while keepingthe temperature at 37° C. During the following 60-minute reperfusionperiod, samples of the perfusate were taken after 3, 5, 10, 15, 30, 45and 60 min for analysis of creatine kinase (CK) activity. Means andstandard deviations for the measured parameters were analyzedstatistically (Dunnett test). The significance limit was p=0.05.

The experiment on rabbit hearts was carried out similarly. Male whiteNew Zealand rabbits (obtained from: Interfauna) were used. The heartswere prepared as described above for the rat model. The perfusionpressure was set at a maximum of 60 mmHg and the flow rate at about 25ml/min. The equilibration time was about 30 min. The substance wasadministered by infusion directly upstream of the heart. 15 min afterstarting the infusion, a 30-minute global ischemia was caused bystopping the flow while maintaining the temperature of the heart. A30-minute reperfusion followed. Perfusate was taken for investigation ofCK activity before administration of the substance, after 15 min and atvarious times (5, 10, 15, 20, 30 min) during the reperfusion. Thefollowing parameters were measured: LVP (mmHg), LVEDP, LVdP/dt, PP(mmHg), HR (pulse rate; beats/min), CK activity (U/min/g heart weight).

c) Animal Model for Acute Kidney Failure

The protective effect of intravenous administration of PARP inhibitors(4 days) on the kidney function of rats with postischemic acute kidneyfailure was investigated.

Male Sprague-Dawley rats (about 330 g at the start of the experiments;breeder: Charles River) were used. 10-15 animals were employed perexperimental group. Administration of active substance/placebo tookplace continuously with an osmotic micropump into the femoral vein.Orbital blood was taken (1.5 ml of whole blood) under inhalationanesthesia with enflurane (Ethrane Abbot, Wiesbaden).

After the initial measurements (blood sample) and determination of theamount of urine excreted in 24 h, the rats were anesthetized(“Nembutal”, pentobarbital sodium, Sanofi CEVA; 50 mg/kg i.p., volumeinjected 1.0 ml/kg) and fastened on a heatable operating table (37° C.).125 IU/kg heparin (Liquemin N, Roche) were administered i.v. into thecaudal vein. The abdominal cavity was opened and the right kidney wasexposed. The branching-off renal artery was exposed and clamped offsuperiorly using bulldog clamps (Diefenbach 38 mm). The left renalartery was likewise exposed and clamped off (superiorly, about half wayto the kidney). During the operation, an osmotic micropump was implantedinto the femoral vein. The intestine was reinserted and the fluid losswas compensated with luke-warm 0.9% NaCl. The animals were covered witha moist cloth and kept warm under red light. After 40 min, theappearance of the kidneys was recorded, and the clamps were removed,first the right then the left. The intestine was put back and 2 drops ofantibiotic (Tardomyocel, Bayer) were added. The abdominal wall wasclosed with sterile cat gut (Ethicon No. 4) and treated once more with 1drop of antibiotic. The epidermis was sutured with sterile Ethibond Exel(Ethicon) No. 3/0, and the suture was sprayed with Nebacetin N(Yamanouchi) wound spray. A tenth of a daily dose of drug/placebo isgiven as i.v. bolus.

Samples and blood were taken for investigating biochemical parameters inthe serum and urine: Na, K, creatinine, protein (only in urine), on days1, 2 and 4 of the experiment. In addition, the feed and waterconsumption, bodyweight and urine volume were recorded. After 14 days,the animals were sacrificed and the kidneys were assessed.

The assessment excluded all animals which died of an infarct during theexperiment or showed an infarct at necropsy on day 14. The creatinineclearance and the fractional sodium excretion were calculated as kidneyfunction parameters, comparing treated animals with control and sham.

d) In Vitro Model for Radiosensitization (Tumor Therapy)

MCF-7-cells (human breast carcinoma) were cultivated in Dulbecco'smodified Eagle's medium with 10% heat-inactivated FCS and 2 mML-glutamine. Cells were seeded out overnight in cell densities of 100,1000 or 10,000 cells per well in a 6-well plate and then exposed toionizing radiation with a dose in the range from 0 to 10 Gy (¹³⁷Cs,Shepard Mark, model I-68A, dose rate 3.28 Gy/min). 10 days after theirradiation, the experiment was assessed, counting colonies with fiftycells as positive.

e) Stroke Model (Focal Cerebral Ischemia; MCA (Middle Cerebral Artery)Occlusion on a Rat)

A focal ischemia was performed by means of cauterisation of the rightdistal MCA on Sprague-Dawley or Long-Evans rats. The rats may be treatedbefore or after the beginning of the MCA occlusion with modulators ofthe proteins of the invention. As a rule, doses of 1-10 mg/kg are chosen(bolus application), optionally followed by a continuous infusion of0.5-5 mg/kg/h.

The rats are anesthetised with halothane in a mixture of 70% nitrogenand 30% oxygen (4% at initial phase and 0.8-1.2% during the operation).The body temperature was permanently measured rectally and was keptconstant at 37.5° C.±0.5° C. by means of a controllable heating blanket.Moreover, arterial blood pressure, arterial pH, (Pa(O₂) and Pa(CO₂) wereoptionally measured by means of a tail vein catheder. Thereafter, thefocal iscehmia was carried out using the method of Chen et al. (Stroke17: 738-743; 1986) or Liu et al. (Am. J. Physiol. 256: H589-593; 1989)by means of continuous cauterisation of the distal part of the rightMCA. When the operation was terminated, the animals were kept in a warmenvironment for a further 24 hours. Then they were killed with the useof CO₂ and decapitated. Their brains were taken, shock-frozen (dry iceor liquid nitrogen) and stored at −80° C. The brains were cut into 0.02mm thick slices and every 20th cut was used for the subsequent analysis.The corresponding cuts are stained with cresyl violet (Nissl staining).Alternatively, TTC (2,3,4-triphenyltetrazoliumchloride) may be used forstaining. The infarct volume may then be analysed under a microscope.For exact quantification, a computer-based image analyzing software maybe used (J. Cereb. Clood Flow Metabol. 10: 290-293; 1990).

f) Septic Shock

Groups of 10 male C57/BL mice (body weight 18-20 g) are treated with LPS(lipopolysaccharide, from E. coli, LD₁₀₀ 20 mg/animal i. v.) plusgalactosamine (20 mg/animal i. v.). the substance to be tested isapplied i. p. or i. v. during three succeeding days (e.g. 1-10 mg/kg),with the first dose being administered 30 minutes after the LPStreatment. The death rate is determined every 12 hours. Alternatively,the substance may also be applied in several doses spread over the days.

g) Determination of Altered Gene Expression in Aging Cells

The aging of cells is simulated by changing the cell culture media fromthe complete medium with a reduced serum concentration and thereafter isanalysed by means of quantitative PCR or Northern Blotting (Linskens etal., Nucleic Acids Res. 1995, 23(16): 3244-51). As typical markers forthe aging of the skin for example collagen or elastin may be used. Humanfibroblasts or fibroblast cell lines are used which simulate the agingof the skin. Modulators of the proteins of the invention are added tothe medium and their effect on the changing of the gene expression isobserved. An increased production of elastin in cells with a reducedaging process caused by means of said modulators may be observed.

1. An isolated and purified-poly(ADP-ribose) polymerase (PARP) homologcomprising human PARP2 (SEQ ID NO: 2).